Abstract Fungal natural products (NPs) have been a preeminent source of medicine and played pivotal roles as pharmaceuticals for the treatment of human diseases. The rapid expansion of fungal genome sequences and the development of bioinformatics tools have enabled the identification of thousands of fungal NP biosynthetic gene clusters (BGCs), thus providing an unprecedented opportunity to discover new fungal NPs. However, the discovery of new bioactive fungal NPs remains challenging, due to difficulties in prioritizing BGCs and genetic manipulations in fungi. In this proposal, we expect to build pipelines to rapidly discover novel bioactive fungal natural products that can serve as the next generation of drug candidates for the treatment of human diseases; to do this, we will apply the CRISPR Cas genome editing technologies and dedicate these tools to the biosynthesis of fungal natural products. To achieve the research goal, our first direction will focus on identifying and characterizing rarely discovered ribosomally synthesized and post?translationally modified peptides (RiPPs) from fungal origins. Due to RiPPs? unique biosynthetic machinery, complex chemical characteristics, and important pharmacological properties, bacterial RiPPs have drawn strong interest from both academia and the pharmaceutical industry. However, only a handful of RiPPs have been identified from fungi, even though fungi is known to be a profilic producer of NPs. By characterizing novel biosynthetic enzymes of known RiPPs and new fungal BGCs identified by bioinformatics analysis, we expect to greatly broaden and deepen our understanding of the biosynthesis of fungal RiPPs and expand the repertoire of novel fungal RiPP NPs. Our second direction will focus on expanding and applying CRISPR?based genome engineering toolkits to characterize biosynthetic gene clusters from filamentous fungi. CRISPR?Cas tools have been successfully demonstrated to be feasible in fungal species but are rarely applied in the investigation of fungal NP biosynthesis. We will develop complementary sets of CRISPR?Cas tools for manipulating fungal biosynthetic gene clusters in both native and heterologous expression hosts. By doing so, we expect to develop a full set of CRISPR gene?editing toolkits to rapidly carry out genetic manipulations to study natural product biosynthesis in filamentous fungi. Together, the two research directions and collaborative research endeavors through BGC characterization, genetic tool advancement, and new bioinformatics algorithm development will build a complete pipeline to significantly increase the repertoire of fungal NPs and analogs, especially fungal RiPPs, making these molecules valuable drug candidates for human therapeutics.